RNA-seq analysis of gene expression changes during pupariation in Bactrocera dorsalis (Hendel) (Diptera: Tephritidae)

Abstract

Background: The oriental fruit fly, Bactrocera dorsalis (Hendel) has been considered to be one of the most important agricultural pest around the world. As a holometabolous insect, larvae must go through a metamorphosis process with dramatic morphological and structural changes to complete their development. To better understand the molecular mechanisms of these changes, RNA-seq of B. dorsalis from wandering stage (WS), late wandering stage (LWS) and white puparium stage (WPS) were performed.

Results: In total, 11,721 transcripts were obtained, out of which 1914 genes (578 up-regulated and 1336 down-regulated) and 2047 genes (655 up-regulated and 1392 down-regulated) were found to be differentially expressed between WS and LWS, as well as between WS and WPS, respectively. Of these DEGs, 1862 and 1996 genes were successfully annotated in various databases. The analysis of RNA-seq data together with qRT-PCR validation indicated that during this transition, the genes in the oxidative phosphorylation pathway, and genes encoding P450s, serine protease inhibitor, and cuticular proteins were down-regulated, while the serine protease genes were up-regulated. Moreover, we found some 20-hydroxyecdysone (20E) biosynthesis and signaling pathway genes had a higher expression in the WS, while the genes responsible for juvenile hormone (JH) synthesis, degradation, signaling and transporter pathways were down-regulated, suggesting these genes might be involved in the process of larval pupariation in B. dorsalis. For the chitinolytic enzymes, the genes encoding chitinases (chitinase 2, chitinase 5, chitinase 8, and chitinase 10) and chitin deacetylase might play the crucial role in the degradation of insect chitin with their expressions significantly increased during the transition. Here, we also found that chitin synthase 1A might be involved in the chitin synthesis of cuticles during the metamorphosis in B. dorsalis.

Conclusions: Significant changes at transcriptional level were identified during the larval pupariation of B. dorsalis. Importantly, we also obtained a vast quantity of RNA-seq data and identified metamorphosis associated genes, which would all help us to better understand the molecular mechanism of metamorphosis process in B. dorsalis.

Keywords: Bactrocera dorsalis; Gene expression; Metamorphosis; Pupariation; RNA-Seq.

Fig. 1

a The morphology of three developmental stages including wandering stage (WS), late wandering stage (LWS), and white puparium stage (WPS) during the pupariation of B. dorsalis. Numbers of differentially expressed genes (DEGs, FDR < 0.01 and |log2 ratio| ≥ 1), the up-regulated genes were represented by a red dot and down-regulated genes by a green dot. b DEGs of WS vs. LWS (c) and WS vs. WPS

Fig. 2

KEGG pathway classification for differentially expressed genes (DEGs) during the pupariation of B. dorsalis. a KEGG pathway classification for DEGs between wandering stage (WS) and late wandering satge. b KEGG classification of DEGs in WS and white puparium stage (WPS)

Fig. 3

KEGG significant enrichment analysis for differentially expressed genes (DEGs). a KEGG significant enrichment analysis for the up-regulated genes between wandering stage (WS) and late wandering stage (LWS). b KEGG significant enrichment analysis for the down-regulated genes that between WS and LWS. c KEGG significant enrichment analysis for the up-regulated genes between WS and white puparium stage (WPS). d KEGG significant enrichment analysis for the down-regulated genes between WS and WPS

Fig. 4

The KEGG pathway of the oxidative phosphorylation pathway responds to pupariation, and genes highlighted in green are enriched and down-regulated. a wandering stage (WS) vs. late wandering stage. b WS vs. white puparium stage

Fig. 5

The expression patterns (FPKM value) of five 20E biosynthesis and seven signaling pathway genes during the pupariation in Bactrocera dorsalis. Three replications were conducted, and the data are presented as mean ± SE. Significant differences among the three treatments were analyzed by one-way analysis. Bars with different letters above them differ significantly at P < 0.05

Fig. 6

Expression of the selected 17 genes by qRT-PCR. a Expression profiles of genes involved in the chitin degradation pathway. b Expression profiles of genes involved in the chitin biosynthesis pathway. The data is presented as mean ± SE of three replications. Stages that are statistically different (P < 0.05) are marked with a different letter (one-way analysis)